Novel rhodamine dyes and conjugates

ABSTRACT

The present invention relates generally to novel rhodamine dyes which upon conjugation with another molecule form single isomeric conjugation products. These novel rhodamine dyes contain only one single functional group on the rhodomine molecule for conjugation so that their conjugation products are single isomeric conjugation products.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. Provisional PatentApplication No. 61/320,571 filed Apr. 2, 2010, the contents of which arcincorporated herein by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

None.

FIELD OF THE INVENTION

The present invention relates generally to novel rhodamine dyes whichupon conjugation with another molecule to form single isomericconjugation products.

BACKGROUND OF THE INVENTION

Rhodamine dyes fluoresce and have been used extensively in research,both as free dye and as conjugates to larger molecules, e.g. proteinsand antibodies (Lee S, McAuliffe D J, Kodama T, Doukas A G, In vivotransdermal delivery using a shock tube, Shock Waves (2000) 10:307-307;Janson L W, Ragsdale K, Luby-Phelps K, Mechanism and size cutoff forsteric exclusion from actin-rich cytoplasmic domains., Biophys J (1996)71:1228-1234; Pu R, Robinson K R, Cytoplasmic calcium gradients andcalmodulin in the early development of the fucoid alga Pelvetiacompressa., J Cell Sci (1998) 111 (Pt 21):3197-3207; Nishiya T, KajitaE, Horinouchi T, Nishimoto A, Miwa S, Distinct roles of TIR and non-TIRregions in the subcellular localization and signaling properties ofMyD88, FEBS Lett (2007) 581:3223-3229; Tanner G A, Sandoval R M, Dunn KW, Two-photon in vivo microscopy of sulfonefluorescein secretion innormal and cystic rat kidneys. Am J. Physiol Renal Physiol (2004)286:F152-F160).

Structurally, rhodamine is a family of related polycyclic flurone dyeswith a xanthene core.

The general structure of rhodamine is as follows:

The amines of rhodamine can be primary amines, secondary amines ortertiary amines

One of the commonly used fluorescent rhodamine dye is sulforhodamine 101which contains a julolidine structure element:

Sulforhodamine 101 contains bi-functional sulfonyl groups as shownbelow:

Sulfonylrhodamine 101 has been used in neurophysiological experimentswhich comprise calcium imaging methods as well as a counterstaining ofastrocytes (Nimmerjahn, A., Kirchhoff, F., Kerr, J. N., Helmchen, F.,Sulforhodamine 101 as a specific marker of astroglia in the neocortex invivo, Nature Methods (2004) 1:31-7).

A sulfonyl chloride derivative of sulforhodamine 101 is sold by SigmaAldrich, Inc. (St. Louis, Mo.) under the trademark Texas Red®. It isused for conjugation with a number of functional groups, especially withprimary amines. Texas Red® fluoresces at about 615 nm with a peakabsorption at 589 nm. Texas Red® is typically available as a mixture oftwo monosulfonyl chlorides with the SO₃ and SO₂Cl groups exchangeable asshown below:

Other rhodamine derivatives have also been disclosed, such as in PCTInt. Appl. (2009), WO 2009108905 A2 20090903; U.S. Pat. Appl. Publ. No.2004054162 A1 20040318; U.S. Pat. No. 5728529; U.S. Pat. No. 5686261;PCT Int. Appl. (1997), WO 9700967 A1 19970109; U.K. Pat. Appl. (1995),GB 2283744 A 19950517; and by Kim etl al. (Kim, T. G.; Castro, J. C.;Loudet, A.; Jiao, J. G.-S.; Hochstrasser, R. M.: Burgess, K.; Topp, M.R., Journal of Physical Chemistry A (2006), 110(1), 20-27).

Although several publications show the possibility of usingbi-functional rhodamine dyes for conjugation, e.g. by selective reactionof one of the sulfonyl chloride groups in Texas Red® (Titus J A,Haugland R, Sharrow S O, Segal D M, Texas Red, a hydrophilic,red-emitting fluorophore for use with fluorescein in dual parameter flowmicrofluorometric and fluorescence microscopic studies, J. Immunol.Methods (1982) 50 (2): 193-204), the possibility of dual reactivitygives difficulties in establishing a reliable process yielding a singleisomer product only, which is shown in FIG. 1 in which a bi-functionalrhodamine dye with two sulfonyl groups reacting with a primary amine toform two conjugation isomeric products. Two isomeric structures insteadof only one in a process gives two major disadvantages: 1) the ratio ofisomers changes from batch to batch with product impacting effects, and2) the regulatory requirements (toxicity data, stability,characterization, etc) will be doubled to cover two substances insteadof one.

The present invention circumvents these difficulties by using novelmono-functional derivatives of rhodamine dye with only one singlefunctional group on the rhodomine molecule for conjugation so that theirconjugation products are single isomeric conjugation products.

These and other aspects and attributes of the present invention will bediscussed with reference to the following drawings and accompanyingspecification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the conjugation of a bi-functional rhodamine dye with amacromolecule to form two isomeric conjugation products;

FIG. 2 shows the conjugation of a mono-functional rhodamine dye with amacromolecule to form only one single isomeric conjugation product;

FIG. 3 shows the formation of the novel rhodamine dye of the presentinvention from 8-hydroxyjulolidine (2 equivalents) and a substitutedbenzaldehyde (1 equivalent) wherein R₁, R₂, R₃, and R₅ can be a H or anygroup;

FIG. 4 shows the general conjugation reaction under the Ugi reactionconditions between a rhodamine dye with a mono-functional group and amacromolecule; and

FIG. 5 is an example of conjugation with the Ugi reaction of amono-functional 2-sulforhodamine (2-SHR) dye having a single functionalprimary amino group with carboxmethylated dextran to form a singleisomeric conjugation product;

FIG. 6 is a UV absorption spectrum of Compound 18 scanning from 200 nmto 800 nm at a scan speed of 400 nm/minute;

FIG. 7 is a fluorescence emission scan of Compound 18;

FIG. 8 is a fluorescence excitation scan of Compound 18;

FIG. 9 is a 3-dimensional fluorescence scan of Compound 18 wherein EM isthe emission wavelength and EX is the excitation wavelength;

FIG. 10 is a UV absorption spectrum of the conjugate of Example 26scanning from 200 nm to 800 nm at a scan speed of 400 nm/minute;

FIG. 11 is a fluorescence emission scan of the conjugate of Example 26;

FIG. 12 is a fluorescence excitation scan of the conjugate of Example26; and

FIG. 13 is a 3-dimensional fluorescence scan of the conjugate of Example26 wherein EM is the emission wavelength and EX is the excitationwavelength.

DETAILED DESCRIPTION OF THE INVENTION

While this invention is susceptible of embodiments in many differentforms, there are specific embodiments which will be described herein indetail with the understanding that the present disclosure is to beconsidered as an exemplification of the principles of the invention andis not intended to limit the invention to the specific embodimentsillustrated.

It will be understood that the chemical structures that are used todefine compounds of the present invention arc each representations ofone of the possible resonance structures that each given structures canhe represented by. Further, it will be understood that by definition,resonance structures are merely a graphical representation used by thoseof skill in the art to represent electron delocalization, and that thepresent disclosure are not limited in any way by showing one particularresonance structure for a given structure.

It will also be understood that the chemical structures that are used todefine compounds of the present invention also include, their structuresin their respective salt forms.

The present invention relates generally to novel rhodamine dyes whichupon conjugation with another molecule to form single isomericconjugation products.

Although several publications show the possibility of usingbi-functional rhodamine dyes for conjugation, e.g. by selective reactionof one of the sulfonyl chloride groups in Texas Red® (Titus J A,Haugland R, Sharrow S O, Segal D M, Texas Red, a hydrophilic,red-emitting fluorophore for use with fluorescein in dual parameter flowmicrofluorometric and fluorescence microscopic studies. J. Immunol.Methods (1982) 50 (2): 193-204), the possibility of dual reactivitygives difficulties in establishing a reliable process yielding a singleisomer product only. As shown in FIG. 1, a bi-functional rhodamine dyewith two sulfonyl groups reacts with a primary amine to form twoconjugation isomeric products. Two isomeric structures instead of onlyone in a process gives two major disadvantages: 1) the ratio of isomerschanges from batch to batch with product impacting effects, and 2) theregulatory requirements (toxicity data, stability, characterization,etc) will be doubled to cover two substances instead of one.

The present invention circumvents these difficulties by using novelmono-functional derivatives of rhodamine dye with only one singlefunctional group on the rhodomine molecule for conjugation so that theirconjugation products arc single isomeric conjugation products asillustrated in FIG. 2. FIG. 2 illustrates an example of the presentinvention in which a rohodamine derivative with a single sulfonyl groupreacts with a primary amine to form only a single isomeric conjugationproduct. What is meant by a “functional group” is that the group issuitable for conjugation. The functional group suitable for conjugationis reactive to another molecule, such as a macromolecule, to form aconjugate via a covalent bond. A rhodamine derivative containing onlyone “functional group” is known as mono-functionalized or amono-functional derivative (such as the mono-sulfonyl rhodamine in FIG.2) which differentiates from rohodamine derivatives containing more thanone “functional groups” such as sulfonyl rhodamine 101 or Texas Red®.Examples of functional groups suitable for conjugation include but arenot limited to amines, isocyanates, isothiocyanates, thiols, carboxylicacids and the like. “Functionalized” herein means that the rhodaminederivative has been derivatised to contain a “functional group”. Anexample is “amino-functionalized” meaning that the functional groupcontains the reactive amino group.

In an embodiment, the novel rhodamine dyes of the present invention havea general structure of:

wherein R1, R2, R3, R4 and R5 can be a H or any group. However, amongR1, R2, R3, R4 and R5, only one of these groups can have a “functionalgroup” so that the rhodamine dye has only one single “functional group”capable of conjugation with another molecule, such as a macromolecule,to form a single conjugation isomeric product. This general structurecan be formed by reacting 8-hydroxyjulodine (2 equivalents) with asubstituted benzaldehyde (1 equivalent) as shown in FIG. 3. The8-hydroxyjulodine and the substituted benzaldehyde can be mixed with 60%aqueous sulfuric acid (11.1 mL/mmol benzaldehyde) and stirred at 150° C.for 24 h under air atmosphere. The reaction mixture can be added to ice(28 g/mmol benzaldehyde), after which 60% NaOH can be carefully added topH6-7 to precipitate the crude product. The crude product can beextracted between dichloromethane (DCM) and water. The organic phase canbe separated, and washed with brine. The organic solvent can be removedand the final product dried by evaporating with ethanol and toluene 5times to give the crude product. Detailed methods for preparing specificexamples of the rhodamine dyes of the present invention are described inExamples below.

Some examples of general structures of mono-functional rhodaminederivatives suitable to form single isomeric conjugation products areshown below with the general formula of 2-sulforhodamine:

-   or the general formula of 4-carboxrhodamine:

-   or the general formula of 3-carboxrhodamine:

-   or the general formula of 4-arylrhodamine:

-   wherein for all the above 4 general formulas. Ar is an aryl group,    and R1 and/or R2 form a spacer with a single functional group on    either R1 or R2 suitable for conjugation with another molecule,    wherein the spacer can be, but is not limited to, hydrogen, alkyl,    aryl, amide, alkyl sulfonamide, alkyl ether, alkyl amide and the    like, or a combination thereof. The alkyl groups mentioned above    preferably have a carbon chain length of from 1 to 20. R1 and R2 can    also be connected to form a cyclic structure, such as but not    limited to the structures shown below:

Although the examples shown above show that the sulfo-group is in the 2position, the carbox-group is in the 3 or 4 position and the aryl-groupis in the 4 poistion, it should be noted that these and other groupscontaining the “functional” group can be in the 2, 3 or 4 position. Theexamples below (Examples 4-24) illustrate the synthesis of some of themembers belonging to the groups shown above. However, the synthesis ofother related compounds with the sulfo-, carbox-, aryl- or any othergroup positioning at any desired position in the novel rhodamine dye ofthe present invention should be obvious to those skilled in the art withthe illustrations from the Examples.

In a preferred embodiment, the novel rhodamine derivatives are in theform of a salt, such as but are not limited to trifluoroacetate,chloride, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate,bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate,salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucaronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzensulfonate and p-toluenesulfonate. In a furtherpreferred embodiment, the salt is trifluoroacetate or chloride. In yetanother preferred embodiment, the salt is a pharmaceutically acceptablesalt.

One of the applications for these novel mono-functional rhodaminederivatives is their ability to conjugate to another molecule, such as amacromolecule. The molecule, such as a macromolecule, when conjugated tothe rhodamine dye can be easily detectable and/or quantifiable. Commonlyused macromolecules herein include but are not limited to polymers,proteins (such as antibodies), dextrans, celluloses, carbohydrates,lipids, nucleic acids (such as DNA and RNA) and the like. Conjugation ofrhodamine dyes with macromolecules and their applications are well knownto those skilled in the art, and has been described in details in thescientific literature, such as disclosed by Titus et al. (Titus J A,Haugland R, Sharrow S O, Segal D M, Texas Red, a hydrophilic,red-emitting fluorophore for use with fluorescein in dual parameter flowmicrofluorometric and fluorescence microscopic studies, J. Immunol.Methods 50 (1982) 2: 193-204) and by Haugland et al. in U.S. Pat. No.5,798,276. Conjugates of rhodamine with macromolecules such asantibodies are readily commercially available such as Human IgG antibodyconjugated with rhodamine from Abeam (Cambridge, Mass.) and variousproteins conjugated with rhodamine dyes from Sigma Aldrich (St. Louis,Mo.). Any synthetic methodology that creates a covalent bond between thefunctional group of the dye and the macromolecule can be used forconjugation. The general conjugation reaction between the rhodamine dyeand a macromolecule under the Ugi reaction conditions is illustrated inFIG. 4, exemplified by the conjugation of the amino-functionalizedsulfonamide dye (Compound 1 shown in Example 6) to carboxymethylateddextran with the Ugi reaction, as shown in FIG. 5.

To illustrate the invention, a number of structures suitable forconjugation have been synthesized (Compounds 1-19) which are shown inthe Examples 6-24 below. Experimental details of further examples ofconjugations are shown with Examples 25-28 below, using, dyes from thesynthesized examples (Compound 18, Compound 3, Compound 15 and Compound16) conjugating with caiboxymethylated-dextrari (CM-dextran).

While the present invention is described in connection with what ispresently considered to be the most practical and preferred embodiments,it should be appreciated that the invention is not limited to thedisclosed embodiments, and is intended to cover various modificationsand equivalent arrangements included within the spirit and scope of theclaims. Modifications and variations in the present invention may bemade without departing from the novel aspects of the invention asdefined in the claims. The appended claims should be construed broadlyand in a manner consistent with the spirit and the scope of theinvention herein.

EXAMPLES

Solvents and reagents are used as received from Labscan (Gliwice,Poland) and Sigma Aldrich (St. Louis, Mo.), respectively. A LiquidChromatography-Mass Spectroscopy (LCMS) system from Agilent Technologies(Santa Clara, Calif.) is used, which consists of a G1379B degasser, aG1312A binary pump, a G1329A autoinjector, a G1316A column oven, aG1365B UV-Vis detector (used to detect absorbance maxima) and a 6110Quadrupole MS detector. High Performance Liquid Chromatography (HPLC)purities are measured with an ACE-C8 column (50×4.6 mm) held at 35° C.and eluted with 10-97% acetonitrile in 0.1% trifluora) acetic acid (TFA)over a 3 minute gradient.

The compounds in the following Examples are named based on Marvin Sketch5.2.6, using Preferred TUPAC naming settings for structure naming.However, other nomenclature systems can be employed to name thesecompounds.

Example 1 Preparation Intermediate 1

The following compound is prepared:

Intermediate 1

A reaction mixture of 8-hydroxyjutolidine (1.1 g, 5.8 mmol) and sodium2-formylbenzene-1 sulfonate (0.6 g, 2.9 mmol) in 60% aqueous H₂SO₄ (10mL) is stirred at 150° C. under an air atmosphere for 2 hours, afterwhich time the starting materials have converted to the expectingproduct completely. pH of the reaction is adjusted to about 7 withaqueous 60% of NaOH, in which procedure the expecting product isprecipitated. The precipitation is filtered and washed with toluene(3×50 mL) and dried under vacuum. The crude product is dissolved in warmethanol (EtOH) and filtered. Insoluble solid is discarded, and thefiltrate is evaporated in vacuo with toluene (3×50 mL), and 1.1 g titlemolecule is obtained with 90% HPLC purity and 73% yield.

Example 2 Preparation of Intermediate 2

The following compound is prepared:

Intermediate 2

8-Mydroxyjulolidine (1.4 g, 7.2 mmol) and 4-formyl benzoic acid (500 mg,3.6 mmol) are mixed with 60% aqueous sulfuric (40 ml) and stirred at150° C. for 24 hours under air atmosphere. The reaction mixture is addedto ice (100 g), after which 60% NaOH is carefully added to pH 6-7,precipitating the crude product. The crude product is extracted betweendichloromethane (DCM) and water. Tire organic phase is separated, andwashed with brine. The organic solvent, is removed and the finalproducts are dried by evaporating with EtOH and toluene 5 times to yield1.1 g product (61% yield). Purity as determined by HPLC is 100%. MS(ESI) [M+]=491.

Example 3 Preparation of Intermediate 3

The following compound is prepared:

Intermediate 3

8-Hydroxyjulolidine (1.4 g, 7.2 mmol) and 3-formyl benzoic acid (500 mg,3.6 mmol) are mixed with 60% aqueous sulfuric (40 mL) and stirred at150° C. for 24 hours under air atmosphere. The reaction mixture is addedto ice (100 g), after which 60% NaOH is carefully added to pH 6-7,precipitating the crude product. The crude product is extracted betweenDCM and water. The organic phase is separated, and washed with brine.The organic solvent is removed and the final products arc dried byevaporating with EtOH and toluene 5 times to yield 1.7 g 3-acid product(94% yield). Purity as determined by HPLC is 95%. MS (ESI) [M+]=491.

Example 4 Preparation of Intermediate 4

The following compound is prepared:

Intermediate 4

8-Hydroxyjulolidine (1.4 g, 7.2 mmol) and 4-bromobenzaldehyde (670 mg,3.6 mmol) arc mixed with 60% aqueous sulfuric (40 mL) and stirred at150° C. for 24 hours under air atmosphere. T he reaction mixture isadded to ice (100 g), after which 60% NaOH is carefully added to pH 6-7,precipitating the crude product and dried under vacuum overnight to 1.12g black solid. The crude product is dissolved in 90/10 CHCl₃/methanol (5mL) and applied on to a column of silica (35×100 mm) and eluted with10-18% methanol in CHCl₃. Pure fractions are pooled and the solvent isevaporated at reduced pressure to 448 mg product.

Example 5 Preparation of Intermediate 5

The following compound is prepared:

Intermediate 5

Intermediate 4 prepared as described in Example 4 (236 mg, 0.45 mmol)and tert-butyl N-{[4-(dihydroxyboranyl)phcnyl]methyl}carbamate (225 mg,0.90 mmol) are transferred to a 50 mL flask with ethanol and 2M K₂CO₃aqueous solution (672 μL, 1.34 mmol) added. The reaction mixture isbriefly degassed and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II). DCM (18 mg,0.022 mmol)) is added, degassing again and stirred under nitrogen atroom temperature. After 50 minutes of stirring, add more[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(11). DCM (13 mg,0.016 mmol) is added and the mixture heated to 65° C. for 1 hour. Thisis followed by evaporation and dissolution in DCM, addition of 4 gsilica and evaporation and silica chromatography through a 12×2.5 cmcolumn, packed with DCM, eluted with 5-20% methanol (MeOH) in DCM andevaporation of pure fraction gave 236 mg golden brown sticky solid with90% HPLC purity.

Example 6 Preparation of a 2-Sulforhodamine Trifluoroacetate (Compound1)

The following compound is prepared:

Compound 3 is16-{2-[(6-aminohexyl)sulfamoyl]phenyl}-3-oxa-9λ⁵,23-diazaheptacyclo[17.7.1.1^(5,9).0^(2.17).0^(4,18).0^(23,27).0^(13.28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium;2,2,2-trifluoroacetate according to the nomenclature system that we useand is prepared as follows. Intermediate 1 (0.66 mmol, 350 mg) isdissolved in DCM (10 mL) and a drop of dimethylformamide (DMF).Oxalylchloride (3.98 mmol, 500 mg) is added and the reaction mixture isstirred at room temperature for one hour. An evolution of gas isimmediately noted. The solvent is evaporated, mixed with toluene (10 mL)and reevaporated, the residue dissolved in DCM (12 mL), cooled in an icebath, and divided into two equal portions. One portion is carefully(under 5 minutes) added to an ice cold solution of hexamethylenediamine(3.06 mmol, 120 mg) in DCM (5 mL) and triethylamine (0.40 mmol, 40 mg)in DCM (5 mL). The dark bluish solutions switch immediately to dark red.Reaction completes within 30 minutes. A part of the crude mixture ispurified on preparative HPLC, ACE-C8 column with a methanol gradient in0.1% TFA in water to give 58 mg (24%) of product as a dark blue coppershimmering glass. Purity as determined by HPLC is 100%. MS (ESI)[M+]=625. Absorbance max is 586 nm.

Example 7 Preparation of a 2-Sulforhodamine Trifluoroacetate (Compound2)

The following compound is prepared:

Compound 2 is16-{2-[3-(aminomethyl)pyrrolidine-1-sulfonyl]phenyl}-3-oxa-9λ⁵,23-diazaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(13,28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium;2,2,2-trifluoroacetate according to the nomenclature system that we useand is prepared as follows. Intermediate 1 (0.66 mmol, 350 mg) isdissolved in DCM (10 mL) and a drop of DMF. Oxalylchloride (3.98 mmol,500 mg) is added and the reaction mixture is stirred at room temperaturefor one hour. An evolution of gas is immediately noted. The solvent isevaporated, mixed with toluene (10 ml) and reevaporated, the residuedissolved in DCM (12 mL), cooled in an ice bath, divided into two equalportions. One portion is carefully (under 5 minutes) added to an icecold solution of tert-butyl N-(pyrrolidin-3-ylmethyl)carbamate (1.06mmol, 90 mg) in DCM (5 mL) and triethylamine (0.40 mmol, 40 mg) in DCM(5 mL). The dark bluish solutions switch immediately to dark red.Reaction completes within 30 minutes. TFA is added (1 mL in ca 2 mL ofDCM), completes deprotection in one hour. A part of the crude ispurified on preparative HPLC, ACE-C8 column with a methanol gradient in0.1% TFA in water to give 95 mg (39%) as a dark blue copper shimmeringglass. Purity as determined by HPLC is 100%. MS (ESI) [M]=605.Absorbance max is 590 nm.

Example 8 Preparation of a 4-carboxrhodamine trifluoroaceate (Compound3)

The following compound is prepared:

Compound 3 is16-{4-[(2-aminoethyl)carbamoyl]phenyl}-3-oxa-9λ⁵,23-diazaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,5).0^(23,27).0^(13,28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium;2,2,2-trifluoroacetate according to the nomenclature system that we useand is prepared as follows. Intermediate2(150 mg, 0.31 mmol) isdissolved in DMF-CH₃CN (1-4, 7 mL). Triethylamine (94 mg, 0.93 mmol) and2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxidesolution in ethyl acetate (592 μL, 0.93 mmol) are added and the mixturestirred at room temperature for 15 minutes. A ⅙-part is taken out andadded to 1,2-diaminoethane (19 mg, 0.31 mmol) and the reaction mixturestirred at room temperature for 1.5 hours. Purification is preformed onpreparative HPLC, ACE-C8 column with a methanol gradient in 0.1% TFA inwater to give 24 mg (72%) as a dark blue black glass. Purity asdetermined by HPLC is 93%. MS (ESI) [M+]=533. Absorbance max is 586 nm.

Example 9 Preparation of a 4-carboxrhodamine trifluoroaceate (Compound4)

The following compound is prepared:

Compound 4 is16-{4-[(3-aminopropyl)carbamoyl]phenyl}-3-oxa-9λ⁵,23-diazabeptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(13,28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium;2,2,2-trifluoroacetate according to the nomenclature system that we useand is prepared using the same procedure as used for preparing Compound3, but with 1,3-diaminopropane replacing 1,2-diaminoethane (23 mg, 0.31mmol) to obtain 25 mg (73% yield) product. Purity as determined by HPLCis 100%. MS (ESI) [M+]=547. Absorbance max is 584 nm.

Example 10 Preparation of a 4-carboxrhodamine trifluoroaceate (Compound5)

The following compound is prepared:

Compound 5 is16-{4-[(2,2-dimethoxyethyl)carbamoyl]phenyl}-3-oxa-9λ⁵,23-diazaheptacyclo[17.7.1.1.^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(13,28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium;2,2,2-trifluoroacetate according to the nomenclature system that we useand is prepared using the same procedure as used for preparing Compound3, but with 2,2-dimethoxyethan-1-amine replacing 1,2-diaminoethane (33mg, 0.31 mmol) to obtain 26 mg (73% yield) product. Purity as determinedby HPLC is 300%. MS (ESI) [M+]=578. Absorbance max is 584 nm.

Example 11 Preparation of a 4-carboxrhodamine trifluoroaceate (Compound6)

The following compound is prepared:

Compound 6 is16-{4-[(6-aminohexyl)carbamoyl]phenyl}-3-oxa-9λ⁵,23-diazaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(13,28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium;2,2,2-trifluoroacetate according to the nomenclature system that we useand is prepared using the same procedure as used for preparing Compound3, but with 1,6-diaminohexane replacing 1,2-diaminoethane (36 mg, 0.31mmol) to obtain 32 mg (88% yield) product. Purity as determined by HPLCis 100%. MS (ESI) [M+]=589. Absorbance max is 586 nm.

Example 12 Preparation of a 4-carboxrhodamine trifluoroaceate (Compound7)

The following compound is prepared:

Compound 7 is16-[4-({[4-(aminomethyl)phenyl]methyl}carbamoyl)phenyl]-3-oxa-9λ⁵,23-diazaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(13,28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium;2,2,2-trifluoroacetate according to the nomenclature system that we useand is prepared using the same procedure as used for preparing Compound3, but with [4-(aminomethyl)phenyl]methanamine replacing1,2-diaminoethane (42 mg, 0.31 mmol) to obtain 36 mg (96% yield)product. Purity as determined by HPLC is 99%. MS (ESI) [M+]=609.Absorbance max is 590 nm.

Example 13 Preparation of a 4-carboxrhodamine trifluoroaceate (Compound8)

The following compound is prepared:

Compound 8 is16-{4-[(4-oxopiperidin-1-yl)carbonyl]phenyl}-3-oxa-9λ⁵,23-diazaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(13,28)]octacosa-1(27),26(17),4,9(28),13,15,18-heptaen-9-ylium;2,2,2-trifluoroacetate according to the nomenclature system that we useand is prepared using the same procedure as used for Compound 3, butwith piperidin-4-one replacing 1,2-diaminoethane (31 mg, 0.31 mmol) toobtain 28 mg (79% yield) product. Purity as determined by HPLC is 82%.MS (ESI) [M+]=572. Absorbance is max 588 nm.

Example 14 Preparation of a 3-carboxrhodamine trifluoroaceate (Compound9)

The following compound is prepared:

Compound 9 is16-{3-[(2-aminoethyl)carbamoyl]phenyl}-3-oxa-9λ⁵,23-diazaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(13,28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium;2,2,2-trifluoroacetate according to the nomenclature system that we useand is prepared using the same procedure as used for Compound 3, butwith Intermediate 3 replacing 1,2-diaminoethane as acid and1,2-diaminoethane (19 mg, 0.31 mmol) as amine to obtain 20 mg (60%yield) product. Purity as determined by HPLC is 100%. MS (ESI) [M+]=533.Absorbance max is 586 nm.

Example 15 Preparation of a 3-carboxrhodamine trifluoroaceate (Compound10)

The following compound is prepared:

Compound 10 is1-{3-[(3-aminopropyl)carbamoyl]phenyl}-3-oxa-9λ⁵,23-diazaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(13,28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium;2,2,2-trifluoroacetate according to the nomenclature system that we useand is prepared using the same procedure as used for preparing Compound9, but with 1,3-diaminopropane replacing 1,2-diaminoethane (23 mg, 0.31mmol) to obtain 19 mg (56% yield) product. Purity as determined by HPLCis 100%. MS (LSI) [M+]=547. Absorbance max is584 nm.

Example 16 Preparation of a 3-carboxrhodamine trifluoroaceate (Compound11)

The following compound is prepared:

Compound 11 is16-{3-[(6-aminohexyl)carbamoyl]phenyl}-3-oxa-9λ⁵,23-diazaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(13,28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium;2,2,2-trifluoroacetate according to the nomenclature system that we useand is prepared using the same procedure as used for preparing Compound9, but with 1,6-diaminohexane replacing 1,2-diaminoethane (36 mg, 0.31mmol) to obtain 27 mg (74% yield) product. Purity as determined by HPLCis 100%. MS (ESI) [M+]=589. Absorbance max is 584 nm.

Example 17 Preparation of a 3-carboxrhodamine trifluoroaceate (Compound12)

The following compound is prepared:

Compound 12 is16-[3-({[4-(aminoethyl)phenyl]methyl}carbamoyl)phenyl]-3-oxa-9λ⁵,23-diazaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(13,28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium;2,2,2-trifluoroacetate according to the nomenclature system that we usearid is prepared using the same procedure is as used for preparingCompound 9, but with [4-(aminomethyl)phenyl]methanamine replacing1,2-diaminoethane (42 mg, 0.31 mmol) to obtain 32 mg (86% yield)product. Purity as determined by HPLC is 100%. MS (ESI) [M+]=609.Absorbance max is 587 nm.

Example 18 Preparation of a 3-carboxrhodamine trifluoroaceate (Cornpound13)

The following compound is prepared:

Compound 13 is16-{3-[(4-oxopiperidin-1-yl)carbonyl]phenyl}-3-oxa-9λ⁵,23-diazaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(13,28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium;2,2,2-trifluoroacetate according to the nomenclature system that we useand is prepared using the same procedure as used for preparing Compound9, but with piperidin-4-one replacing 1,2-diaminoethane (31 mg, 0.31mmol) to obtain 34 mg (96% yield) product. Purity as determined by HPLCis 80%. MS (ESI) [M+]=572. Absorbance max is 584 nm.

Example 19 Preparation of a 3-carboxrhodamine trifluoroaceate (Compound14)

The following compound is prepared:

Compound 14 is16-{3-[(2,2-dimethoxyethyl)carbamoyl]phenyl}-3-oxa-9λ⁵,23-diazaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(13,28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium;2,2,2-trifluoroacetate according to the nomenclature system that we useand is prepared using the same procedure is as used for preparingCompound 9, but with 2,2-dimethoxyethan-1-amine replacing1,2-diaminoethane (33 mg, 0.31 mmol) to obtain 29 mg (81% yield)product. Purity as determined by HPLC is 98%. MS (ESI) [M+]=578.Absorbance max is 588 nm.

Example 20 Preparation of a 4-carboxrhodamine trifluoroaceate (Compound15)

The following compound is prepared:

Compound 15 is16-{4-[4-(aminomethyl)phenyl]phenyl}-3-oxa-9λ⁵,23-diazaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(13,28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium;2,2,2-trifluoroacetate according to the nomenclature system that we useand is prepared as follows. Intermediate 5(236 mg, 0.35 mmol) isdissolved in 4 mL DCM/TFA (3/1). After 30 minutes, the solvent is blownoff with air. The crude product is purified on an ACE-C8 (150×30 mm)column eluting with 20-100% methanol in 0.1% TFA. Pure fractions areevaporated to 115 mg (% yield) deep blue/red solid with a green tinge.Purity as determined by HPLC is 98%. MS (ESI) [M+]=552. Absorbance maxis 578 nm.

Example 21 Preparation of a 4-carboxrhodamine trifluoroaceate (Compound16)

The following compound is prepared:

Compound 16 is16-[4-(2-amino-4-methylphenyl)phenyl]-3-oxa-9λ⁵,23-diazaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(13,28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium;2,2,2-trifluoroacetate according to the nomenclature system that we useand is prepared as follows. Intermediate 4 (152 mg, 0.29 mmol) and5-methyl-2-(4,4,5,5-tetramethyl)-(1,3,2-dioxaborolan-2-yl)-phenylamine(135 mg, 0.58 mmol) are dissolved in ethanol (10 mL) and treated with 2MK₂CO₃ (434 μL, 0.87 mmol) and[1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II)DCM (11 mg,0.015 mmol). The reaction mixture is heated at 65° C. for 1 hour. Thecrude product is filtered through celite and purified on an ACE-C8(150×30 mm) column eluting with 60-100% methanol in 0.1% TFA,evaporating pure fractions to 80 mg (50% yield) deep blue/red solid witha green tinge. Purity as determined by HPLC is 90%. MS (ESI) [M+]=552.Absorbance max is 580 nm.

Example 22 Preparation of a 4-carboxrhodamine trifluoroaceate (Compound17)

The following compound is prepared:

Compound 17 is16-[4-(4-acetylphenyl)phenyl]-3-oxa-9λ⁵,23-diazaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(13,28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium;2,2,2-trifluoroacetate according to the nomenclature system that we useand is prepared as follows. Intermediate 4(152 mg, 0.29 mmol) and4-ethanone phenyl boronic acid (95 mg, 0.58 mmol) are dissolved inethanol (10 mL) and treated with 2M K₂CO₃(434 μL, 0.87 mmol) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(11). DCM (11 mg,0.015 mmol). The reaction mixture is heated at 65° C. for 1 hour. Thecrude product is filtered through celite and purified on an ACB C8(150×30 mm) column eluting with 60-100% methanol in 0.1% TFA,evaporating pure fractions to 54 mg (33% yield) deep blue/red solid witha green tinge. Purity as determined by HPLC is 92%. MS (ESI) [M+]=565.Absorbance max is 580 nm.

Example 23 Preparation, Purification and Crystallization of a2-sulforhodamine dichloride (Compound 18)

The following compound is prepared:

Compound 18 is16-{2-[(6-aminohexyl)sulfamoyl]phenyl}-3-oxa-9λ⁵,23-diazaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,15).0^(23,27).0^(13,28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium;dichloride according to the nomenclture system that we use and isprepared as follows.

Synthetic Step 1: A mixture of 8-hydroxyjulolidine (200 g) and sodium2-formylbenzene-1-sulfonate (110 g) is added to 1.8 L of 60% H2SO4aqueous pre-warmed at 150° C. and stirred for 4 hours. After thereaction is finished monitored by LC-MS, the reaction mixture is cooledto 0° C. 60% of sodium hydroxide (aqueous) is added slowly, until pHvalue of the reaction mixture to 2 (product is precipitated). Celite(800 g) is added to the reaction mixture with precipitated raw productand the reaction mixture is filtered. The resulting solid with celite iswashed with toluene (3×500 mL) and added to anhydrous ethanol (4 L) andheated with stiring at 60° C. for 0.5 hour. The reaction mixture then isfiltered. The filtrate is concentrated under vacuum and thenco-evaporated with toluene to get rid of traces of water and to getcrude Rhodamine sulfonic acid intermediate (270 g) as a dark blue solid.

Synthetic Step 2: Crude Rhodamine sulfonic acid intermediate from Step 1(100 g, 0.19 mol) is dissolved in a mixture of solvents dichloromethane(500 mL) and DMF (13.9 g). The reaction solution is cooled to 0° C. andoxalyl chloride (48.1 g, 0.379 mol) is added dropwise. The reactionmixture is stirred for additional 2 hours at 0° C. The reaction mixtureis then concentrated in vacuum and to the resulting residue is fouriterations of adding toulene (100 mL) and evaporation performed. Thecrude Rhodamine sulfonic acid chloride intermediate is used directly innext step after drying under reduced pressure for 6 h.

Synthetic Step 3: A solution of hexane-1,6-diamine (176 g. 1.52 mol) indichloromethane (800 ml) is cooled to 0° C. A solution of all crudeRhodamine sulfonic acid chloride intermediate obtained in Step 2 indichloromethane (500 mL) is added dropwise with continued cooling to 0°C. The reaction mixture is stirred for an additional 4 hours at 0° C.After controlling with thin layer chromatography (TLC) that the reactionis complete, the reaction mixture is quenched by a filtration throughcelite-paper, and evaporating the filtrate in vacuum four times withadditions of toluene (150 mL) each time. The resulting solid is driedunder reduced pressure.

Purification: Purification by chromatography through silica gel iscarried out using a mixture of solvents dichloromethane and MeOH(gradient from 15:1 to 1:1), saturated with HCl gas. A second silica gelchromatography yields 12.5 g of the 2-sulforhodamine dichloride(Compound 18) as a grecn-black solid (purity >98% by HPLC). The totalyield for 3 all steps is 11.0%.

Crystallization: 2.5 g of the 2-sulforhodamine dichloride (Compound 18)of approx 88% purity is dissolved in ethanol (approx 50 mL) and ethylacetate (approx 150 mL) is added, which precipitates a sticky solid. Thesupernatant is treated with silica gel (5 g) and filtered. The silica isleached with 1 M HCl (200 mL), the resulting purple solution is thenused to dissolve the sticky solid that is precipitated with ethylacetate in a previous step. The solution is heated close to boiling andtreated with warm brine (400 mL, aq NaCl saturated at room temperature).The solution is allowed to cool to room temperature and minorprecipitation is observed. pH is adjusted from −0.2 to +0.3 by carefuladdition of solid NaHCO₃. The mixture is heated to boiling, a cooledaliquot of the solution is analyzed to pH 0.0. Solid NaHCO₃ is added tothe warm solution until a cooled sample showed pH 0.1. The suspension isallowed to cool to room temperature, after two days the supernatant isanalyzed to pH 0.0. Solid NaHCO₃ is carefully added to the mixturewithout any more precipitation being observed (use a red lamp!). Thesolid is separated with centrifugation and the supernatant discarded (pH0.0). The solid is suspended in the same volume of 20% aq NaCl that isused in the previous precipitation and centrifuged, discarding thesupernatant (pH 0.5). The solid is again suspended in an equal volume,centrifuged and the supernatant discarded (pH 0.55). The secondsupernatant is not colorless and the washing procedure halted. The solidis dried in vacuum oven to 1.8 g of golden-green material, 96% puritywith Syntagon's HPLC method. The above crystallization process can beused with an acetate salt of the 2-sulforhodamine (such as thetrifluoroaceate salt as in Compound 1) instead of the dichioride salt ofCompound 18.

FIG. 6 is a UV absorption spectrum of Compound 18 scanning from 200 nmto 800 nm at a scan speed of 400 nm/minute. The maximum UV absorption isat 586 nm. FIG. 7 is a fluorescence emission scan and FIG. 8 is afluorescence excitation scan of Compound 18. FIG. 9 is a 3-dimensionalfluorescence scan of Compound 18 wherein EM is the emission wavelengthand EX is the excitation wavelength. The Excitation (max), which is themaximum absorbance wavelength, is 566 nm and the Emission (max), whichis the wavelength with maximum emission intensity, is 618 nm.

Example 24 Rearrangement of Compound 18 to form an isomer (Compound 19)

The following compound is prepared:

Compound 18 can undergo a re-arrangement to form an isomer Compound 19,which can be used like its parent compound Compound 18 and the othernovel rhodamine dyes of the present invention in forming conjugates withother molecules, such as macromolecules, to form single isomericconjugation products. Compound 19 is16-[N-(6-azaniumylhexyl)benzenesulfonamido]-3-oxa-9λ⁵,23-diazaheptacyclo[17.7.1.1^(5,9).0^(2,17).0^(4,l5).0^(23,27).0^(13,28)]octacosa-1(27),2(17),4,9(28),13,15,18-heptaen-9-ylium;dichloride according to the nomenclature system that we use and isprepared as follows. Compound 18 as the dichloride salt (1.16 g, 1.6mmol) is dissolved in methanol (30 mL). Aqueous sodium hydroxide (1.0 M,16 mL) is added drop-wise to the 2-SHR solution at room temperature. Theresulting colorless solution is stirred for 30 min at room temperatureand then evaporated with ethanol (10 mL) and toluene (10 mL) to acolorless solid. The solid is dissolved in methanol (20 mL) and aqueoushydrochloric acid (1.0 M, 16 mL) is added, which reforms the deep redcolor. The mixture is evaporated and the solid residue purified withsilica chromatography, eluting with methanol (10%-12.5%) in chloroformcontaining 0.1% concentrated hydrochloric acid. Selected pure fractionsare evaporated with ethanol and toluene, drying at high vacuum overnightresults in 120 mg 2-SHR-iso (11% yield) as a metallic green solid.Purity as determined by HPLC is 97.8%. MS (ESI) [M+]=625. Absorbance maxis 586 nm.

Similarly, Compound 1 or any other salt of the 2-sulforhodamine canundergo the same re-arrangement as Compound 18 to form the correspondingisomeric product with the corresponding salt.

Example 25 Synthesis of conjugate with Compound 15 andcarboxymethylated-dextran (CM-dextran)

To a solution of Compound 15 (Mw: 665.77 g/mol, n: 0.038 mmol, m: 25 mg)dissolved in dimethyl sulfoxide (DMSO) (3 ml) is added cold acetaldehydefrom a stock solution (118 μl) with stirring. After 15 minutes asolution of CM-dextran 150 (300 mg) in distilled water (3 ml) is addedwith rapid stirring, followed by cyclohexyl-isonitrile (Mw: 109.1 g/mol,n: 0.21 mmol, in: 22.9 mg, δ:0.878 g ml, V: 26.5 μl). The pH is adjustedto 5.9 with a few drops of 1M aq. HCl. The reaction mixture is left withstirring for four hours.

After four hours the pH has increased to 6.02. Ethanolamine (Mw: 61.08g/mol, n: 3.34 mmol, m: 0.204 g, δ: 1.02, V: 200 μl) is added and thereaction is left for 90 minutes with stirring.

Ninety minutes after the addition of ethanolamine the pH has increasedto 11.2. After addition of saturated sodium chloride (0.5 ml), thereaction mixture is slowly poured in to ethanol (96%, 50 ml) with rapidstirring where after the precipitated blue solid is allowed to settleovernight. The supernatant is decanted and the residue is filtered on aglass filter funnel (p3). The precipitate is washed with Ethanol (3×10ml) and filtered. The product is reprecipitated until free fromunreacted dye. It is dried, in vacuo at 60° C. for 15 hours. Yield is225 mg.

Excitation (max) is 587 nm; Emission(max) is 608.

Example 26 Synthesis of conjugate with Compound 18 with CM-dextran

To a solution of Compound 18 (chloride salt, Mw: 697.7 g/mol, n: 0.136mmol, m: 95.4 mg) dissolved in DMSO (14 mL) is added cold acetaldehydefrom a stock solution (391 μL) with stirring. After 15 minutes asolution of CM-dextran 150 (1.8 g) in distilled water (19.2 mL) is addedwith rapid stirring, followed by cyclohexyl-isonitrile (Mw: 109.1 g/mol,n: 1.4 mmol, m: 152.6 mg, 8: 0.878 g/mL, V: 172 μl). The pH is adjustedto 5 with a few drops of 1M aq. HCl. The reaction mixture is left withstirring overnight.

Ethanolamine (Mw: 61.08 g/mol, n: 6.64 mmol, m: 0.40 g, δ: 1.02, V: 400μL) is added and the reaction is left for 60 minutes with stirring.

Ninety minutes after the addition of ethanolamine the pH has increasedto 11.2. After addition of saturated sodium chloride (0.5 ml), thereaction mixture is slowly poured in to ethanol (96%, 50 ml) with rapidstirring where after the precipitated blue solid is allowed to settleovernight.

The supernatant is decanted and the residue is filtered on a glassfilter tunnel (p3). The precipitate is washed with ethanol (3×10 ml) andfiltered. The product is reprecipitated until free from unreacted dye.It is dried in vacuo at 60° C. for 15 hours. Yield is 1.3 g.

FIG. 10 is a UV absorption spectrum of the conjugate scanning from 200nm to 800 nm at a scan speed of 400 nm/minute. The maximum UV absorptionis at 589.5 nm. FIG. 11 is a fluorescence emission scan and FIG. 12 is afluorescence excitation scan of the conjugate. FIG. 13 is a3-dimensional fluorescence scan of the conjugate wherein EM is theemission wavelength and EX is the excitation wavelength.

Excitation (max) is 589 nm; Emission(max) is 608 nm.

Example 27 Synthesis of conjugate with Compound 3 with CM-dextran

To a solution of Compound 3 (TFA salt, Mw: 646.7 g/mol. n: 0.037 mmol,m: 24 mg) dissolved in DMSO (3 mL) is added cold acetaldehyde from astock solution (118 μL) with stirring. After 15 minutes a solution ofCM-dextran 150 (300 mg) in distilled water (3 mL) is added with rapidstirring, followed by cyclohexyl-isonitrile (Mw: 109.1 g/mol, n: 0.21mmol, m: 22.9 mg, δ: 0.878 g/mL, V: 26.5 μL). The pH is adjusted to 5with a few drops of 1M aq. HCl. The reaction mixture is left withstirring for 4 hours.

Ethanolamine (Mw: 61.08 g/mol, n: 6.64 mmol, m: 0.20 g, δ: 1.02, V: 200μL) is added and the reaction is left for 60 minutes with stirring. Thereaction product, after addition of saturated sodium chloride (0.5 ml),is slowly poured into ethanol (96%, 50 ml) with rapid stirringwhereafter the precipitated blue solid is allowed to settle overnight.

The supernatant is decanted and the residue is filtered on a glassfilter funnel (p3). The precipitate is washed with ethanol (3×10 ml) andfiltered. The product is reprecipitated until free from unreacted dye.It is dried in vacuo at 60° C. for 15 hours. Yield is 339 mg.

Excitation (max) is 588 nm; Emission(max) is 609 nm.

Example 28 Synthesis of conjugate with Compound 16 with CM-dextran

To a solution of Compound 16 (TFA salt, Mw: 665.7 g/mol, n: 0.038 mmol,m: 25 mg) dissolved in DMSO (3 mL) is added cold acetaldehyde from astock solution (118 μL) with stirring. After 15 minutes a solution ofCM-dextran 150 (300 mg) in distilled water (3 mL) is added with rapidstirring, followed by cyclohexyl-isonitrile (Mw: 109.1 g/mol, n: 0.21mmol, m: 22.9 mg, δ: 0.878 g/mL, V: 26.5 μL). The pH is adjusted to 5with a few drops of 1M aq. HCl. The reaction mixture is left withstirring for 4 hours.

Ethanolamine (Mw: 61.08 g/mol, n: 6.64 mmol, m: 0.20 g, δ: 1.02, V: 200μL) is added and the reaction is left for 60 minutes with stirring. Thereaction product, after addition of saturated sodium chloride (0.5 ml),is slowly poured into ethanol (96%, 50 ml) with rapid stirring whereafter the precipitated blue solid is allowed to settle overnight. Thesupernatant is decanted and the residue is filtered on a glass filterfunnel (p3). The precipitate is washed with ethanol (3×10 ml) andfiltered. The product is reprecipitated until free from unreacted dye.It is dried in vacuo at 60° C. for 15 hours. Yield is 167 mg.

Excitation (max) is 585 nm; Emission(max) is 606 nm.

REFERENCES

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3. Pu R, Robinson K R, Cytoplasmic calcium gradients and calmodulin inthe early development of the fucoid alga Pelvetia compressa., J Cell Sci(1998) 111 (Pt 21):3197-3207

4. Nishiya T, Kajita E, Horinouchi T, Nishimoto A, Miwa S, Distinctroles of TIR and non-TIR regions in the subcellular localization andsignaling properties of MyD88, FEBS Lett (2007) 581:3223-3229

5. Tanner G A, Sandoval R M, Dunn K W, Two-photon in vivo microscopy ofsulfonefluorescein secretion in normal and cystic rat kidneys, Am JPhysiol Renal Physiol (2004) 286:F152-F160

6. Titus J A, Haugland R, Sharrow S O, Segal D M, Texas Red, ahydrophilic, red-emitting fluorophore for use with fluorescein in dualparameter flow microfluorometric and fluorescence microscopic studies,J. Immunol. Methods (1982) 50 (2): 193-204

7. Nimmerjahn, A., Kirchhoff, F., Kerr, J. N., Helmchen, P.,Sulforhodamine 101 as a specific marker of astroglia in the neocortex invivo, Nature Methods (2004) 1:31-7

8. Kim, T. G.; Castro, J. C; Loudet, A.; Jiao, J. G.-S.; Hochstrasser,R. M.; Burgess, K.; Topp, M. R ., Journal of Physical Chemistry A(2006), 110(1), 20-27

1-119. (canceled)
 120. A conjugate of the formula A-B, wherein A is amolecule covalently linked to a compound B, the compound B comprising arhodamine dye of the structure

wherein R1, R2, R3, R4 or R5 comprises a functional group, wherein: R5is independently H,

R4 is independently H,

R3 is independently H,

R2 is independently H or

and R1 is H, provided that only one of R2, R3, R4 and R5 is not H,wherein Ar is an aryl group, or a pharmaceutically acceptable salt,optical isomer, racemate, diastereomer, or enantiomer.
 121. Theconjugate of claim 120, wherein the functional group of R1, R2, R3, R4or R5 is conjugated to the molecule A.
 122. The conjugate of claim 120,wherein R₆ or R₇ is conjugated to the molecule A.
 123. The conjugate ofclaim 120, wherein the functional group is selected from the groupconsisting of an amine, an alcohol, an isocyanate, an isothiocyanate, athiol and a carboxylic acid, optionally wherein the functional group isan amine.
 124. The conjugate of claim 120, wherein the molecule A is amacromolecule.
 125. The conjugate of claim 124, wherein themacromolecule is selected from the group consisting of a polymer, aprotein, a polysaccharide, a polysaccharide derivative, a lipid and anucleic acid.
 126. The conjugate of claim 125, wherein the protein is anantibody.
 127. The conjugate of claim 125, wherein the nucleic acid is aDNA or an RNA.
 128. The conjugate of claim 125, wherein thepolysaccharide comprises dextran, optionally wherein the dextran iscarboxymethylated-dextran.
 129. The conjugate of claim 120, wherein thepharmaceutically acceptable salt is selected from the group consistingof trifluoroacetate, chloride, hydrochloride, hydrobromide, hydroiodide,nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate,acetate, lactate, salicylate, citrate, tartrate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, fortmate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzensulfonate andp-toluenesulfonate, optionally wherein the pharmaceutically acceptablesalt is trifluoroacetate or chloride.
 130. The conjugate of claim 120,wherein R₆ and R₇ form a spacer.
 131. The conjugate of claim 130,wherein the spacer is selected from the group consisting of an alkyl, anaryl, an amide, an alkyl sulfonamide, an alkyl ether, an alkyl amine andan alkyl amide, optionally wherein the spacer is an alkyl amine. 132.The conjugate of claim 131, wherein the alkyl has a carbon chain lengthof 1 to
 20. 133. The conjugate of claim 120, wherein R₆ and R₇ form acyclic structure.
 134. The conjugate of claim 133, wherein the cyclicstructure is


135. The conjugate of claim 120, wherein R1, R2, R4 and R5 are H and R3is


136. The conjugate of claim 120, wherein R1, R2, R4 and R5 are H and R3is


137. The conjugate of claim 120, wherein R1, R2, R4 and R5 are H and R3is


138. The conjugate of claim 120, wherein R1, R2, R3 and R4 are H, R5 is

wherein one of R₆ and R₇ is

and the other of R₆ and R₇ is H.
 139. A composition comprising:

or a pharmaceutically acceptable salt, optical isomer, racemate,diastereomer, or enantiomer.
 140. A process for preparing the conjugateof claim
 120. 141. The process of claim 140, wherein the processcomprises treating an intermediate with a catalyst.
 142. The process ofclaim 141, wherein the intermediate is selected from the groupconsisting of:


143. The process of claim 141, wherein the catalyst is1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (DCM). 144.The process of claim 143, comprising adding the molecule A to theintermediate and the catalyst to yield the conjugate of formula A-B.145. The process of claim 144, wherein the process is performed in thepresence of cyclohexile-isonitrile.
 146. A method for performingfluorescence microscopy, the method comprising detecting the conjugateof claim 120.